1. Field of the Invention
The present invention relates to a method for the enzymatic production of 2-hydroxy-2-methyl carboxylic acids from 3-hydroxy carboxylic acids, where a 3-hydroxy carboxylic acid is produced in an aqueous reaction solution and/or is added to this reaction solution and is incubated. The aqueous reaction solution comprises a unit having 3-hydroxy-carboxylate-CoA mutase activity which has both 3-hydroxy-carbonyl-CoA ester-producing and 3-hydroxy-carbonyl-CoA ester-isomerizing activity and converts the 3-hydroxy carboxylic acid into the corresponding 2-hydroxy-2-methyl carboxylic acid which is isolated as acid or in the form of its salts. In a preferred embodiment, the unit having 3-hydroxy-carboxylate-CoA mutase activity comprises an isolated cobalamin-dependent mutase and where appropriate a 3-hydroxy-carbonyl-CoA ester-producing enzyme or enzyme system or is a microorganism including them. The invention preferably relates to a biotechnological process for producing 2-hydroxy-2-methyl carboxylic acids, where microorganisms which have the desired mutase activity are cultured in an aqueous system with the aid of simple natural products and intracellularly formed 3-hydroxy-carbonyl-CoA esters are converted into the corresponding 2-hydroxy-2-methyl carboxylic acids. The invention likewise encompasses the production of unsaturated 2-methyl carboxylic acids, where the 2-hydroxy-2-methyl carboxylic acids obtained are converted by dehydration into the corresponding unsaturated 2-methyl carboxylic acids (methacrylic acid and higher homologs).
In a preferred embodiment of the invention, the 3-hydroxy-carbonyl-CoA thioester-producing and 3-hydroxy-carbonyl-CoA thioester-isomerizing microorganism used is the strain HCM-10 (DSM 18028).
2. Discussion of the Background
Methacrylic acid and homologous unsaturated 2-methyl carboxylic acids are widely used in the production of acrylic glass sheets, injection-molded products, coatings and many other products.
A plurality of processes for the production of methacrylic acid and its homologs have been disclosed. However, the vast majority of the commercial production worldwide is based on a method of hydrolyzing the amide sulfates of methacrylic acid and its homologs, which are produced from the corresponding 2-hydroxy nitriles (W. Bauer, “Metharylic acid and derivatives”, in: Ullmann's Encyclopedia of Industrial Chemistry, 5th edition, editors: B. Elvers, S. Hawkins, G. Schulz, VCH, New York, 1990, Vol. A16, pp. 441-452; A. W. Gross, J. C. Dobson, “Methacrylic acid and derivatives”, in Kirk-Othmer Encyclopedia of Chemical Technology, 4th edition, editors: J. I. Kroschwitz, M. Howe-Grant, John Wiley & Sons, New York, 1995, Vol. 16, pp. 474-506). This method requires, for example, about 1.6 kg of sulfuric acid for the production of 1 kg of methacrylic acid. For this reason, alternative methods for the commercial production of methacrylic acid without the requirement of recovering the sulfuric acid (and the high energy costs associated therewith) would be advantageous.
U.S. Pat. No. 3,666,805 and U.S. Pat. No. 5,225,594 have disclosed the chemical conversion of 2-hydroxy isobutyric acid to methacrylic acid. This comprises dehydrating 2-hydroxy isobutyric acid by using metal oxides, metal hydroxides, ion exchange resins, alumina, silicon dioxide, amines, phosphines, alkali metal alkoxides and alkali metal carboxylates. Usual reaction temperatures are between 160° C. and 250° C. This method made possible methacrylic acid yields of up to 96%.
An alternative method for the production of methacrylic acid and its homologs is based on the hydrolysis of 2-hydroxy nitriles to the corresponding 2-hydroxy-2-methyl carboxylic acids, utilizing nitrile-hydrolyzing enzymes. The latter are nitrilase or a combination of nitrile hydratase and amidase (A. Banerjee, R. Sharma, U. C. Banerjee, 2002, “The nitrile-degrading enzymes: current status and future prospects”, Appl. Microbiol. Biotechnol., 60:33-44). This method is protected by a plurality of patents (U.S. Pat. No. 6,582,943 B1). A severe disadvantage of this method is the instability of the nitriles in the neutral pH range required for an efficient nitrile-hydrolyzing enzyme activity. The decomposition of the nitriles in the reaction mixture results in accumulation of ketones and cyanides, both of which inhibit the nitrile-hydrolyzing enzyme activities.
A general disadvantage of both methods, i.e. of the currently dominating method based on amide sulfates and of the enzymatic nitrile-hydrolyzing method, is the need for 2-hydroxy nitriles. The latter must first be prepared from environmentally harmful reactants, namely ketones and cyanide.
For this reason, methods for the production of methacrylic acid and its homologs, which are based on simple environmentally benign reactants, would be advantageous.